It is generally believed that shutting down the kinase activity of BCR-ABL by imatinib will completely inhibit its functions, leading to inactivation of its downstream signaling pathways and cure of the disease. Imatinib is highly effective at treating human Philadelphia chromosome-positive (Ph ؉ ) chronic myeloid leukemia (CML) in chronic phase but not Ph ؉ B cell acute lymphoblastic leukemia (B-ALL) and CML blast crisis. We find that SRC kinases activated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells, suggesting that imatinib does not inactivate all BCR-ABLactivated signaling pathways. This SRC pathway is essential for leukemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Inhibition of both SRC and BCR-ABL kinase activities by dasatinib affords complete B-ALL remission. However, curing B-ALL and CML mice requires killing leukemic stem cells insensitive to both imatinib and dasatinib. Besides BCR-ABL and SRC kinases, stem cell pathways must be targeted for curative therapy of Ph ؉ leukemia.dasatinib ͉ imatinib ͉ SRC kinases

Glucocorticoid hormones, including dexamethasone, induce apoptosis in lymphocytes and consequently are used clinically as chemotherapeutic agents in many hematologic malignancies. Dexamethasone also induces autophagy in lymphocytes, although the mechanism is not fully elucidated. Through gene expression analysis, we found that dexamethasone induces the expression of a gene encoding a stress response protein variously referred to as Dig2, RTP801, or REDD1. This protein is reported to inhibit mammalian target of rapamycin (mTOR) signaling. Because autophagy is one outcome of mTOR inhibition, we investigated the hypothesis that Dig2/RTP801/REDD1 elevation contributes to autophagy induction in dexamethasone-treated lymphocytes. In support of this hypothesis, RNAimediated suppression of Dig2/RTP801/REDD1 reduces mTOR inhibition and autophagy in glucocorticoid-treated lymphocytes. We observed similar results in Dig2/Rtp801/Redd1 knock-out murine thymocytes treated with dexamethasone. Dig2/RTP801/REDD1 knockdown also leads to increased levels of dexamethasone-induced cell death, suggesting that Dig2/ RTP801/REDD1-mediated autophagy promotes cell survival. Collectively, these findings demonstrate for the first time that elevation of Dig2/RTP801/REDD1 contributes to the induction of autophagy.

Glucocorticosteroid hormones, including prednisone and dexamethasone (Dex), have been used to treat lymphoid malignancies for many years because they readily induce apoptosis in immature lymphocytes lacking Bcl-2. However, elevated expression of the anti-apoptotic protein Bcl-2 inhibits apoptosis and contributes to glucocorticoid resistance. Using the Bcl-2-negative WEHI7.2 lymphoma line as an experimental model, we found that Dex not only induces apoptosis but also induces autophagy. The caspase inhibitor Z-VAD-fmk inhibited apoptosis but not autophagy in Dex-treated cells. Bcl-2 overexpression inhibited Dex-induced apoptosis even more potently than Z-VAD-fmk and, contrary to previous reports, Bcl-2 neither interacted with Beclin-1 nor inhibited autophagy. Rather, Bcl-2 overexpression facilitated detection of Dex-induced autophagy by both steady state methods and flux measurements, ostensibly due to apoptosis inhibition. Autophagy contributed to prolonged survival of Bcl-2-positive lymphoma cells following Dex treatment, as survival was reduced when autophagy was inhibited by 3-methyladenine. These findings emphasize the important interplay between apoptosis and autophagy and suggest a novel mechanism by which Bcl-2, which is frequently elevated in lymphoid malignancies, contributes to glucocorticoid resistance and survival of lymphoma cells.

Since publication of this article, we have become aware of an error in the experimental protocol to visualize LAMP1 in cells by immunofluorescence. Specifically, the wrong antibodies were used, so assessments of LAMP1 distribution are not reliable. We did additional experiments, including analysis of the distribution of LAMP2, which is found in the same compartments as LAMP1. We observed no co-localization of LAMP2 and Stbd1. However, the primary conclusion of the study, the link between Stbd1 and glycogen metabolism, is unaffected, as is our hypothesis that Stbd1 anchors glycogen to membranes and may be involved in its localization and trafficking within the cell. The grant information footnote should read as follows. This work was supported, in whole or in part, by National Institutes of Health Grants R01 CA42755 and CA85804 (to C. W. D.), R01 AG031903 (to S. M.), T32 HL007147 (to S. S. and J. K. M.), and T32 GM007250 (to J. K. M.). The "Acknowledgments" should read as follows. The Dig2/RTP801 knock-out mice were obtained from Quark Pharmaceuticals, Inc., for whom they were exclusively generated at Lexicon. We thank Tamotsu Yoshimori and Noboru Mizushima for providing LC3 cDNA and Mark Jackson for suggestions regarding lentiviral shRNA. This work was supported in part by an Ohio Center for Innovative Immunosuppressive Therapeutics grant, which maintains the spinning disk confocal microscope in the Morphology Core Facility of the Department of Dermatology, Case Western Reserve University.THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 45, p. 39673, November 11, 2011 © 2011 ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.

The calcium ion, a major intracellular second messenger, is a known mediator of apoptosis and is regulated by the antiapoptotic protein Bcl-2. A paper by Høyer-Hasen et al. (2007) in the current issue of Molecular Cell indicates that calcium also mediates the induction of macroautophagy in a Bcl-2 regulated fashion and identifies a signaling pathway through which calcium exerts its action. These intriguing findings provoke speculation as to how a cell decides to undergo either apoptosis or macroautophagy in response to calcium signals.

The lacI gene of E. coli has been a highly useful target for studies of mutagenesis, particularly for analysis of the specificity (spectrum) of mutations generated under a variety of conditions and in various genetic backgrounds. The gene encodes the repressor of the lac operon, and lacI-defective mutants displaying constitutive expression of the operon are readily selected. DNA sequencing of the lacI mutants has often been confined to the N-terminal region of the protein, as it presents a conveniently short target with a high density of detectably mutable sites. Mutants in this region are easily selected due to their dominance in a genetic complementation test (lacId mutants). A potential complication in these studies is that constitutive expression of lac may also arise due to mutations in the lac operator (lacO mutants). Under some conditions, for example when analyzing spontaneous mutations, lacO mutants can comprise a very high fraction of the constitutive mutants due to a strong base-substitution hotspot in the lac operator. Such mutational hot spots diminish the return of the sequencing effort and do not yield significant new information. For this reason, a procedure to eliminate the lacO mutants prior to DNA sequencing is desirable. Here, we report a simple method that allows screening out of lacO mutants. This method is based on the lack of resistance of lacO mutants to kanamycin under conditions when the kan gene is expressed from a plasmid under control of the lac promoter-operator (lacPO). We show data validating the new approach with sets of known lacId and lacO mutants, and further apply it to the generation of a new collection of spontaneous mutations, where lacO mutants have historically been a significant contributor.

A report on research that explicates three models of pedagogical practice that underpin and characterize inquiry instruction in a course-based research experience.

The ABL kinase inhibitor imatinib mesylate is the preferred treatment for Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) in chronic phase, but is less effective in CML blast crisis or Ph+ B-cell acute lymphoblastic leukemia (B-ALL). We have shown that SRC kinases are required for BCR-ABL-induced B-ALL but not CML (Hu et al., Nat. Genet., 36:453, 2004), suggesting that SRC kinases may also serve as therapeutic targets in addition to BCR-ABL for B-ALL. To test this idea, we compared the activities of Abl-selective imatinib with BMS-354825, a potent dual inhibitor of SRC and ABL kinases, using the BCR-ABL mutant P210T315I resistant to inhibition by both drugs (Shah et al, Science, 16:399, 2004). We treated P210T315I-expressing pre-B cells with BMS-354825 in vitro. While BMS-354825 did not reduce phosphorylation of P210T315I, it inhibited phosphorylation of SRC kinases at concentrations as low as 25 nM, confirming that BMS-354825 is a potent SRC inhibitor in cells. Moreover, BMS-354825 inhibited proliferation and induced apoptosis of the cells, suggesting that inhibition of SRC kinases alone may suffice in inhibiting the growth of BCR-ABL inhibitor-insensitive leukemic cells. To test whether SRC inhibition was sufficient to exert a therapeutic effect on P210T315I-induced B-ALL, donor bone marrow cells from BALB/c mice were transduced with P210T315I-IRES-GFP retrovirus, followed by transplantation into BALB/c recipients. 8 days after the transplantation, BMS-354825 was given orally twice a day at a dose of 30 mg/kg for 6 days, followed by switching to 10 mg/kg for 3 days and then back to 30mg/kg. Imatinib was given orally twice a day at a dose of 100mg/kg. At day 17 post BMT, the mean peripheral blood (PB) GFP+/B220+ cell count (/ml) was: 1.8x107 for placebo; 0.7x107 for BMS-354825; and 3.0x107 for imatinib. So far (30 days post BMT), 50% (3/6) of placebo-treated and 69% (9/13) of imatinib-treated mice died, whereas all BMS-354825-treated mice are alive. These results suggested that SRC kinases could serve as therapeutic targets for B-ALL although targeting SRC alone may not cure the disease. Similarly, targeting BCR-ABL alone with imatinib could not cure B-ALL. To test dual-target therapy of B-ALL with BMS-354825, we treated mice with B-ALL induced by wild type BCR-ABL. Compared to placebo- and imatinib-treated mice at day 17 post BMT, GFP+/B220+ cells in BMS-354825-treated mice were no longer detectable while PB GFP+/B220+ cells for placebo- and imatinib-treated groups were 3.6x107and 2.4x107/ml, respectively. Although this therapeutic experiment is still ongoing, complete eradication of leukemic cells in BMS-354825-treated mice demonstrates that blockade of both SRC kinases and BCR-ABL cured B-ALL. To understand how SRC kinases function in B-leukemic cells, we looked for SRC downstream signaling molecules using mice deficient for SRC kinases LYN, HCK or FGR. The activation of AKT and STAT5 but not ERK and JNK was down-regulated in leukemic cells lacking LYN, HCK and FGR. The lack of HCK or FGR but not LYN was responsible for their down-regulation. Together, these results indicate that SRC kinases are valuable therapeutic targets for Ph+ B-ALL, and that simultaneous inhibition of SRC and BCR-ABL kinases and their downstream pathways may be beneficial to Ph+ B-ALL patients.